Ahnak functions as a tumor suppressor via modulation of TGFβ/Smad signaling pathway

Lee, I. H.; Sohn, Minji; Lim, Ho-Jin; Yoon, Sang-Hoon; Oh, Hyung Jung; Shin, Sujin; Shin, Jae Hoon; Oh, Seung Hyun; Kim, Jeewon; Lee, D. K.; Noh, Dong‐Young; Bae, Duk Soo; Seong, Je Kyung; Bae, Yun Soo

doi:10.1038/onc.2014.69

Cited by 114 publications

(128 citation statements)

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“…Specifically, nuclear localization of Ahnak in complex with phospho-Smad3 and binding to promoter of c-Myc were strongly enhanced in response to TGF-␤, indicating that Ahnak downregulates the expression of c-Myc as Smad3 target genes. Consistently, Ahnak-deficient MEF cells show a significant up-regulation of c-Myc expression (17).…”

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confidence: 59%

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Regulation of c-Myc Expression by Ahnak Promotes Induced Pluripotent Stem Cell Generation

Lim

Kim

Park

et al. 2016

Journal of Biological Chemistry

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It has been well established that the so-called Yamanaka factors, Oct4 (octamer-binding transcription factor 4), Sox2 (SRY (sex determining region Y)-box 2), Klf4 (Kruppel-like factor 4), and c-Myc, together stimulate the generation of induced pluripotent stem cells (iPSC) 3 from diverse types of somatic cells including embryonic fibroblast cells (1-5). Oct4 belongs to the family of POU transcriptional factors and binds to an octamer sequence motif, AGTCAAAT, leading to expression of target genes in developing germ cells (6, 7). Sox2 is a member of Sox transcriptional factor family containing the high mobility group domain and is known to be required for maintenance of diverse types of stem cells throughout life (8). KLF4 is a member of Kruppel-like factor transcriptional factor family that binds to CACCC sequence regulating gene expression during embryonic development (9, 10). c-Myc is a well known basic helixloop-helix transcriptional factor that plays important roles in cell cycle regulation, cellular transformation, and maintenance of pluripotency (11,12).Ahnak is an exceptionally large protein (ϳ700 kDa) and has an extended central region composed of 36 repeat units that serve as a scaffolding motif for interaction of phospholipase C-␥ with PKC in regulation of cell proliferation and migration (13-16). Moreover, the central repeated unit is known to bind to R-Smad protein in response to TGF-␤ and mediate down-regulation of c-Myc expression and cell growth retardation. Specifically, nuclear localization of Ahnak in complex with phospho-Smad3 and binding to promoter of c-Myc were strongly enhanced in response to TGF-␤, indicating that Ahnak downregulates the expression of c-Myc as Smad3 target genes. Consistently, Ahnak-deficient MEF cells show a significant up-regulation of c-Myc expression (17).There have been efforts to exclude c-Myc from the mix of ectopically expressed transcription factors for iPSC generation mainly because of its potential transforming activity. Exclusion of c-Myc, however, led to a dramatic reduction in efficiency (18 -20). Therefore, developing a method to efficiently utilize endogenous c-Myc activity for iPSC generation could represent an important progress. Here, we present that Ahnak knock-out MEF or wild type MEF expressing shRNA specific to Ahnak can be converted to iPSC at a high efficiency without infection of c-Myc retrovirus. Further dissection of Ahnak pathway may provide novel strategies for regulation of iPSC generation and reducing the potential danger from cellular transformation.

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confidence: 59%

“…have previously demonstrated that Ahnak plays an inhibitory role in c-Myc expression through TGF␤-Smad3 signaling cascade in epithelial cells (17). To evaluate whether Ahnak regulates c-Myc expression in embryonic fibroblasts, we examined Ahnak knock-out (Ahnak Ϫ/Ϫ ) MEF.…”

Section: Mutually Exclusive Expression Of Ahnak and C-myc-wementioning

confidence: 99%

Regulation of c-Myc Expression by Ahnak Promotes Induced Pluripotent Stem Cell Generation

Lim

Kim

Park

et al. 2016

Journal of Biological Chemistry

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“…The precise function of PDAP1 is unclear, but it has been reported to be highly up-regulated in the secretome of neoplastic gastric epithelial cells (58), a context in which SMYD2 activity was recently characterized (26). Finally, AHNAK and AHNAK2, which are uniquely and extensively directly mono-methylated by SMYD2, appear to possess diverse functionality (reviewed in (59)) with roles ranging from cell adhesion (60), cell signaling (61,62), and tumor cell migration and invasion (63). Whether the extensive mono-methylation of the AHNAK and AHNAK2 CRUs by SMYD2, as well as the individual sites we identified in other SMYD2 substrates, regulates the function of these and other proteins remains to be determined.…”

Section: Discussionmentioning

confidence: 99%

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

Olsen

Cao

Han

et al. 2016

Molecular & Cellular Proteomics

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The significance of non-histone lysine methylation in cell biology and human disease is an emerging area of research exploration. The development of small molecule inhibitors that selectively and potently target enzymes that catalyze the addition of methyl-groups to lysine residues, such as the protein lysine mono-methyltransferase SMYD2, is an active area of drug discovery. Critical to the accurate assessment of biological function is the ability to identify target enzyme substrates and to define enzyme substrate specificity within the context of the cell. Here, using stable isotopic labeling with amino acids in cell culture (SILAC) coupled with immunoaffinity enrichment of mono-methyl-lysine (Kme1) peptides and mass spectrometry, we report a comprehensive, large-scale proteomic study of lysine mono-methylation, comprising a total of 1032 Kme1 sites in esophageal squamous cell carcinoma (ESCC) cells and 1861 Kme1 sites in ESCC cells overexpressing SMYD2. Among these Kme1 sites is a subset of 35 found to be potently down-regulated by both shRNA-mediated knockdown of SMYD2 and LLY-507, a selective small molecule inhibitor of SMYD2. In addition, we report specific protein sequence motifs enriched in Kme1 sites that are directly regulated by endogenous SMYD2 activity, revealing that SMYD2 substrate specificity is more diverse than expected. We further show direct activity of SMYD2 toward BTF3-K2, PDAP1-K126 as well as numerous sites within the repetitive units of two unique and exceptionally large proteins, AHNAK and AHNAK2. Collectively, our findings provide quantitative insights into the cellular activity and substrate recognition of SMYD2 as well as the global landscape and regulation of protein mono-methylation. Protein lysine methyltransferases (PKMTs) 1 catalyze the sequence-specific transfer of one, two, or three methyl groups to the side chains of lysine residues (1-4). In addition to the extensively studied lysine methylation on histones, PKMTs can modify non-histone proteins (3,(5)(6)(7)(8)). An increasing number of non-histone proteins have been reported as PKMT substrates, and, as a result, potential roles for the dysregulation of non-histone lysine methylation in cancer development and progression have been proposed (9). The majority of PKMT substrates have been identified based on biochemical methylation assays using recombinant enzyme and substrate, followed by cell-based assays typically requiring overexpression of enzyme and/or substrate to maximize signal detection (10 -13). However, it is difficult to discern whether these substrates are bona fide physiological substrates of endogenous PKMT activity. With the development of PKMT-targeted drugs emerging as a key area of drug discovery (14 -18), unbiased and quantitative methods enabling the comprehensive identification of histone and non-histone substrates in cells are critical to clarifying the cell-relevant substrates of these enzymes and to more accurately understand the functions of non-histone methylation.Progressing from targeted biochemical...

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“…The majority of biotinylated proteins in AsPC-1 cells were identified as putative K-Ras-interacting partners in transfected HEK293T cells. Noteworthy abundant AsPC-1 hits included the giant, putative tumor suppressor protein AHNAK (67), but also multiple cell surface signaling proteins, including EGFR, integrin β1, mucin 13, ephrin and its receptor, and CD44. These results are discussed below.…”

Section: Identification Of Cellular K-ras-interacting Proteinsmentioning

confidence: 99%

Analysis of K-Ras Interactions by Biotin Ligase Tagging

Ritchie

Mack

Harper

et al. 2017

CGP

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Ahnak functions as a tumor suppressor via modulation of TGFβ/Smad signaling pathway

Cited by 114 publications

References 35 publications

Regulation of c-Myc Expression by Ahnak Promotes Induced Pluripotent Stem Cell Generation

Regulation of c-Myc Expression by Ahnak Promotes Induced Pluripotent Stem Cell Generation

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

Analysis of K-Ras Interactions by Biotin Ligase Tagging

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